Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
Infection with the Human Immunodeficiency Virus (HIV), if left untreated, almost always leads to death of the infected person. HIV infects the CD4+ T-cells and leads to a decline in the number of CD4+ T-cells in the infected person. When CD4+ T-cell numbers decline below a critical level, cell-mediated immunity is effectively lost, and infections with a variety of opportunistic microbes appear, resulting in Acquired Immunodeficiency Syndrome (AIDS). Because the HIV-infected person can no longer defend against these opportunistic infections, the patient will ultimately succumb to one of these infections.
Infection by HIV is mediated by the envelope glycoprotein (Env). Env forms a heterotrimer composed of the receptor binding subunit gp120 and the HIV membrane anchored fusion protein subunit gp41 (see FIG. 1). Entry into host cells is mediated by gp120 interaction with CD4 that triggers a conformational change allowing subsequent interaction with a cellular co-receptor, principally CCR5 or CXCR4 (Dalgleish et al. (1984) Nature 312: 763-767; Klatzmann et al. (1984) Nature 312: 767-768; Moore et al. (1997) Curr Opin Immunol 9: 551-562; Clapham & McKnight (2002) J Gen Virol 83: 1809-1829). CCR5 is the predominant co-receptor used, but an altered use of CCR5 is selected for during progressive HIV infection. Association of gp120 with the co-receptor induces additional conformational changes in gp41, which in turn promote mixing of the membrane lipids, ultimately facilitating fusion of the viral and cellular membranes (Weissenhorn et al. (1997) Nature 387: 426-430; Chan et al. (1997) Cell 89: 263-273; Weissenhorn et al. (2007) FEBS Lett 581: 2150-2155). Once the virus has entered the T-cells, the virus hijacks the replication machinery of the T-cell to produce additional copies of HIV thereby furthering the infection.
Currently there is no cure available for HIV/AIDS. However, HIV infected persons can suppress replication of the virus through a variety of anti-viral treatment options. Current treatment for HIV infection consists of anti-retroviral therapy, or ART. ART consists of the administration of a cocktail of multiple anti-viral compounds. However, because HIV readily mutates the virus often becomes resistant to one or more compounds in the ART cocktail. In addition, ART is associated with a number of side effects. While anti-retroviral adherence is the second strongest predictor of progression to AIDS and death, after CD4 count, incomplete adherence to ART is common in all groups of treated individuals. The average rate of adherence to ART is approximately 70%, despite the fact that long-term viral suppression requires near-perfect adherence. The resulting virologic treatment failure diminishes the potential for long-term clinical success and increases the risk of drug resistance.
New therapies to treat HIV infection are needed therefore.
Next to the small-molecule anti-HIV compounds, neutralizing antibodies have been engineered and tested. However, antibodies are necessarily directed against the exterior of the virus. Indeed, Env is a main target for entry inhibitors (Matthews et al. (2004) Nat Rev Drug Discov 3: 215-225) and most neutralizing antibodies are directed against gp120 or gp41 (Sattentau Q (2008) Curr Opin HIV AIDS 3: 368-374). Also in this case a crucial problem in HIV vaccine research is the generation of cross-subtype neutralizing antibodies, which is due to the fact that HIV employs a number of strategies to evade the immune response. This includes highly variable gp120 regions, a carbohydrate shield (Wyatt et al. (1998) Nature 393: 705-711) and conformational masking of the receptor binding site (Kwong et al. (2002) Nature 420: 678-682).
Nanobodies directed against various HIV-1 proteins have been described (Hinz et al. 2010 PLoS ONE 5:e10482; McCoy et al. 2014 Retrovirology 11:83; Vercruysse et al. 2010 JBC 285:21768-21780; Bouchet et al., 2011 Blood 117:3559-3568). However, various Nanobodies were directed against intra-cellular HIV proteins, necessitating intracellular expression of the Nanobody. Moreover, in none of the cases, the development of resistance by HIV against the Nanobody has been addressed.
Interestingly, the options for HIV to “mutate around” therapies directed at blocking cell entry appear to be more limited. Indeed, in a study using ibalizumab, an anti-CD4 monoclonal antibody, resistance was developed but the resistant isolates remained dependent on CD4 for viral entry, suggesting that resistance did not develop through the use of alternative receptors (cf. Bruno & Jacobson 2010 J Antimicrob Chemother 65:1839-1841). Nevertheless, also in this case resistance against ibalizumab developed eventually (cf. Fessel et al., 2011 Antiviral Res 92:484-487).
PRO140 is a fully humanized IgG4 monoclonal antibody directed against the co-receptor CCR5. PRO140 blocks the HIV R5 subtype entry into T-cells by masking the required co-receptor CCR5. In short term studies, resistance against PRO140 has not been observed. However, the potential development of resistance in long term studies has not been addressed. PRO140 does not prevent the usage of the CXCR4 co-receptor. For instance, in up to 40 to 50% of individuals infected with B-HIV, progression to late stages of infection is associated with a switch in co-receptor specificity, with emergence of X4 (CXCR4) or R5X4 (CCR5/CXCR4) viral variants (Bjorndal et al. J Virol 1997, 71(10):7478-7487; Connor et al. J Exp Med 1997, 185(4):621-628). The emergence of CXCR4-using HIV viruses is associated with rapid CD4+ T-cell decline and progression from chronic to advanced stages of HIV infection.